Recognition of lipid A variants by the TLR4-MD-2 receptor complex

Abstract

Lipopolysaccharide (LPS) is a component of the outer membrane of almost all Gram-negative bacteria and consists of lipid A, core sugars, and O-antigen. LPS is recognized by Toll-like receptor 4 (TLR4) and MD-2 on host innate immune cells and can signal to activate the transcription factor NFκB, leading to the production of pro-inflammatory cytokines that initiate and shape the adaptive immune response. Most of what is known about how LPS is recognized by the TLR4-MD-2 receptor complex on animal cells has been studied using Escherichia coli lipid A, which is a strong agonist of TLR4 signaling. Recent work from several groups, including our own, has shown that several important pathogenic bacteria can modify their LPS or lipid A molecules in ways that significantly alter TLR4 signaling to NFκB. Thus, it has been hypothesized that expression of lipid A variants is one mechanism by which pathogens modulate or evade the host immune response. Additionally, several key differences in the amino acid sequences of human and mouse TLR4-MD-2 receptors have been shown to alter the ability to recognize these variations in lipid A, suggesting a host-specific effect on the immune response to these pathogens. In this review, we provide an overview of lipid A variants from several human pathogens, how the basic structure of lipid A is recognized by mouse and human TLR4-MD-2 receptor complexes, as well as how alteration of this pattern affects its recognition by TLR4 and impacts the downstream immune response.

Keywords: LPS; TLR4; innate immunity; lipid A; signaling.

Figure 1

Schematic of the basic structure of lipopolysaccharide. LPS consists of three regions: from the bottom, lipid A (chair structure indicates di-glucosamine headgroup, red circles indicate phosphate groups, squiggly lines indicate acyl chains), core sugars, and O-antigen, which consists of repeating units (denoted in brackets, with an “n”) of oligosaccharides.

Figure 2

Simplified diagram of signaling by LPS on host cells. LPS is extracted from bacterial membranes by LPS binding protein (LBP) in serum, passed to CD14, then transferred to MD-2 and TLR4, which form a complex on the cell surface. LPS-induced receptor dimerization is followed by recruitment of the adaptor proteins TIRAP and MyD88, which promotes activation of the transcription factor NFκB. The TLR4-MD-2 complex can also be internalized, which recruits a different set of adaptor proteins, TRIF and TRAM, promoting activation of IRF3 and production of type I interferons, as well as delayed activation of NFκB.

Figure 3

Chemical structure of lipid A molecules. (A) Hexa-acylated lipid A from E. coli. Red numbers indicate carbon numbering. (B) Hexa-acylated lipid A from Y. pestis grown at 27°C. (C) Tetra-acylated lipid A from Y. pestis grown at 37°C. (D) Tetra-acylated lipid IVA, a biosynthetic precursor of hexa-acylated lipid A. (E) The synthetic molecule Eritoran. (F) Monophosphoryl lipid A (MPLA). (G) Glucosamine-modified lipid A from B. pertussis (strain BP338, a Tohama I derivative).

Figure 4

Simplified diagram of TLR4-MD-2 receptor complex dimerization upon ligation of hexa-acyl lipid A. The amphipathic lipid A molecule consists of negatively-charged (indicated by a circle with a “−” sign in it) phosphate groups and hydrophobic fatty acyl chains. The fatty acyl chains sit inside the “binding pocket” structure of the MD-2 co-receptor protein, which is lined with hydrophobic amino acid residues, while the phosphate groups are engaged by charged amino acids in MD-2 at the mouth of the pocket. In the next part of the figure, we show lipid A inside the MD-2 pocket and also in contact with TLR4, with which it forms a 1:1 complex. Upon lipid A binding, this TLR4-MD-2 complex dimerizes with a second complex. From this position, lipid A engages the TLR4 molecule in the second complex (TLR4^*) at two main interfaces: the first is mediated by interaction of the 6th acyl chain of lipid A (which is shown sticking out of the pocket) with uncharged amino acids (cloud labeled “neutral”) on TLR4^*, the second is mediated by interaction of the negatively-charged 1-phosphate (1-PO₄) on lipid A with positively-charged amino acid residues (cloud labeled “+”) on TLR4^*.